The B cell response to antigen is regulated by a variety of co-receptors that convey information to the B cell about the quality of the antigen and the status of the ongoing immune response. Our progress using high resolution live cell imaging to delineate the very early antigen driven events in B cell activation has provided a new context in which the impact of coreceptors can be evaluated. Using high resolution fluorescence resonance energy transfer (FRET) coupled with total internal reflection microscopy (TIRFM) and single molecule tracking we provided evidence for an ordered process that occurs within seconds to minutes of the BCR binding antigen. Antigen bound BCRs form immobile clusters that then grow in size by molecular trapping. The clusters perturb the local lipid environment causing lipid rafts to coalesce around the BCR clusters. As a consequence of the membrane perturbation the first kinase in the pathway, Lyn, that is tethered to the membrane by raft lipids is brought into close molecular proximity to the BCR clusters. Simultaneously, Lyn phosphorylates the Ig alpha beta cytoplasmic domain of the BCR and the Ig alpha beta chains undergo a conformational change from a closed to an open form. Syk is recruited to the phosphorylated BCR and the signaling cascades are triggered. Over the last year we characterized the effect of the inhibitory receptor FcgammaRIIB engagement on the activation of human naive and MBCs. Using high-resolution imaging we showed that human MBCs are more robust than naive B cells at each step in the initiation of BCR signaling, including interrogation of antigen containing membranes, formation of sub-microscopic BCR oligomers and recruitment and activation of signaling-associated kinases. Despite their robust response to antigen, MBCs remain highly sensitive to FcgammaRIIB-mediated inhibition. These observations are important for understanding the regulation of memory antibody responses. We also demonstrated in human B cells that the rapid accumulation of BCRs at the interface of the B cell on antigen-containing bilayer triggers the independent accumulation of phosphorylated CD19 accompanied by the accumulation and colocalization of phosphorylated PI3K, a key component in the BCR-CD19 signaling pathway. Remarkably, coligation of the BCR and FcgammaRIIB blocked the accumulation of the BCR, and phosphorylated CD19 and PI3K. These results suggest an additional facet of the inhibition of BCR signaling by the FcgammaRIIB. We will continue these studies in collaboration with Dr. Ezio Bonvini and his colleagues at Macrogenics. Dr. Bonvinis group has developed bi-functional antibodies coined DARTS that allow the dimerization of the BCR and CD19 or the BCR and FcgammaRIIB. The BCR-FcgammaRIIB DARTS are showing efficacy in blocking B cell activation in autoimmune disease and killing BCR-dependent B cell tumors. We are exploring the effect of these DARTS on BCR signaling using high resolution live cell imaging. Over the last year we have made progress in establishing super-high resolution imaging to describe, at the 10-50 nm level, the spatial relationship between the BCR, CD19 and FcgammaRIIB in resting human B cells and in B cells in which the BCR is ligated alone by Ag or Fcgamma is ligated by immune complexes. We are also characterizing the spatial distribution of these receptors on naive human IgM expressing B cells and in IgG-expressing memory B cells.